Annarita Patrizi

Neuroligin-4 is localized to glycinergic postsynapses and regulates inhibition in the retina

Neuroligins (NL1–NL4) are postsynaptic adhesion proteins that control the maturation and function of synapses in the central nervous system (CNS). Loss-of-function mutations in NL4 are linked to rare forms of monogenic heritable autism, but its localization and function are unknown. Using the retina as a model system, we show that NL4 is preferentially localized to glycinergic postsynapses and that the loss of NL4 is accompanied by a reduced number of glycine receptors mediating fast glycinergic transmission. Accordingly, NL4-deficient ganglion cells exhibit slower glycinergic miniature postsynaptic currents and subtle alterations in their stimulus-coding efficacy, and inhibition within the NL4-deficient retinal network is altered as assessed by electroretinogram recordings. These data indicate that NL4 shapes network activity and information processing in the retina by modulating glycinergic inhibition. Importantly, NL4 is also targeted to inhibitory synapses in other areas of the CNS, such as the thalamus, colliculi, brainstem, and spinal cord, and forms complexes with the inhibitory postsynapse proteins gephyrin and collybistin in vivo, indicating that NL4 is an important component of glycinergic postsynapses.

Complex Role of Collybistin and Gephyrin in GABAA Receptor Clustering

Gephyrin and collybistin are key components of GABAA receptor (GABAAR) clustering. Nonetheless, resolving the molecular interactions between the plethora of GABAAR subunits and these clustering proteins is a significant challenge. We report a direct interaction of GABAAR α2 and α3 subunit intracellular M3–M4 domain (but not α1, α4, α5, α6, β1–3, or γ1–3) with gephyrin. Curiously, GABAAR α2, but not α3, binds to both gephyrin and collybistin using overlapping sites. The reciprocal binding sites on gephyrin for collybistin and GABAAR α2 also overlap at the start of the gephyrin E domain. This suggests that although GABAAR α3 interacts with gephyrin, GABAAR α2, collybistin, and gephyrin form a trimeric complex. In support of this proposal, tri-hybrid interactions between GABAAR α2 and collybistin or GABAAR α2 and gephyrin are strengthened in the presence of gephyrin or collybistin, respectively. Collybistin and gephyrin also compete for binding to GABAAR α2 in co-immunoprecipitation experiments and co-localize in transfected cells in both intracellular and submembrane aggregates. Interestingly, GABAAR α2 is capable of “activating ” collybistin isoforms harboring the regulatory SH3 domain, enabling targeting of gephyrin to the submembrane aggregates. The GABAAR α2-collybistin interaction was disrupted by a pathogenic mutation in the collybistin SH3 domain (p.G55A) that causes X-linked intellectual disability and seizures by disrupting GABAAR and gephyrin clustering. Because immunohistochemistry in retina revealed a preferential co-localization of collybistin with α2 subunit containing GABAARs, but not GlyRs or other GABAAR subtypes, we propose that the collybistin-gephyrin complex has an intimate role in the clustering of GABAARs containing the α2 subunit.

Synapse formation and clustering of neuroligin-2 in the absence of GABAA receptors

GABAergic synapses are crucial for brain function, but the mechanisms underlying inhibitory synaptogenesis are unclear. Here, we show that postnatal Purkinje cells (PCs) of GABAAα1 knockout (KO) mice express transiently the α3 subunit, leading to the assembly of functional GABAA receptors and initial normal formation of inhibitory synapses, that are retained until adulthood. Subsequently, down-regulation of the α3 subunit causes a complete loss of GABAergic postsynaptic currents, resulting in a decreased rate of inhibitory synaptogenesis and formation of mismatched synapses between GABAergic axons and PC spines. Notably, the postsynaptic adhesion molecule neuroligin-2 (NL2) is correctly targeted to inhibitory synapses lacking GABAA receptors and the scaffold molecule gephyrin, but is absent from mismatched synapses, despite innervation by GABAergic axons. Our data indicate that GABAA receptors are dispensable for synapse formation and maintenance and for targeting NL2 to inhibitory synapses. However, GABAergic signaling appears to be crucial for activity-dependent regulation of synapse density during neuronal maturation.

Sensory experience regulates cortical inhibition by inducing IGF1 in VIP neurons

Inhibitory neurons regulate the adaptation of neural circuits to sensory experience, but the molecular mechanisms by which experience controls the connectivity between different types of inhibitory neuron to regulate cortical plasticity are largely unknown. Here we show that exposure of dark-housed mice to light induces a gene program in cortical vasoactive intestinal peptide (VIP)-expressing neurons that is markedly distinct from that induced in excitatory neurons and other subtypes of inhibitory neuron. We identify Igf1 as one of several activity-regulated genes that are specific to VIP neurons, and demonstrate that IGF1 functions cell-autonomously in VIP neurons to increase inhibitory synaptic input onto these neurons. Our findings further suggest that in cortical VIP neurons, experience-dependent gene transcription regulates visual acuity by activating the expression of IGF1, thus promoting the inhibition of disinhibitory neurons and affecting inhibition onto cortical pyramidal neurons.

Quantitative organization of GABAergic synapses in the molecular layer of the mouse cerebellar cortex

In the cerebellar cortex, interneurons of the molecular layer (stellate and basket cells) provide GABAergic input to Purkinje cells, as well as to each other and possibly to other interneurons. GABAergic inhibition in the molecular layer has mainly been investigated at the interneuron to Purkinje cell synapse. In this study, we used complementary subtractive strategies to quantitatively assess the ratio of GABAergic synapses on Purkinje cell dendrites versus those on interneurons. We generated a mouse model in which the GABAA receptor α1 subunit (GABAARα1) was selectively removed from Purkinje cells using the Cre/loxP system. Deletion of the α1 subunit resulted in a complete loss of GABAAR aggregates from Purkinje cells, allowing us to determine the density of GABAAR clusters in interneurons. In a complementary approach, we determined the density of GABA synapses impinging on Purkinje cells using α-dystroglycan as a specific marker of inhibitory postsynaptic sites. Combining these inverse approaches, we found that synapses received by interneurons represent approximately 40% of all GABAergic synapses in the molecular layer. Notably, this proportion was stable during postnatal development, indicating synchronized synaptogenesis. Based on the pure quantity of GABAergic synapses onto interneurons, we propose that mutual inhibition must play an important, yet largely neglected, computational role in the cerebellar cortex.

Understanding the Molecular Diversity of GABAergic Synapses

GABAergic synapses exhibit a high degree of subcellular and molecular specialization, which contrasts with their apparent simplicity in ultrastructural appearance. Indeed, when observed in the electron microscope, GABAergic synapses fit in the symmetric, or Gray’s type II category, being characterized by a relatively simple postsynaptic specialization. The inhibitory postsynaptic density cannot be readily isolated, and progress in understanding its molecular composition has lagged behind that of excitatory synapses. However, recent studies have brought significant progress in the identification of new synaptic proteins, revealing an unexpected complexity in the molecular machinery that regulates GABAergic synaptogenesis. In this article, we provide an overview of the molecular diversity of GABAergic synapses, and we consider how synapse specificity may be encoded by selective trans-synaptic interactions between pre- and postsynaptic adhesion molecules and secreted factors that reside in the synaptic cleft. We also discuss the importance of developing cataloguing tools that could be used to decipher the molecular diversity of synapses and to predict alterations of inhibitory transmission in the course of neurological diseases.

Synaptogenesis in the cerebellar cortex: Differential regulation of gephyrin and GABAA receptors at somatic and dendritic synapses of Purkinje cells

In rodent cerebellar cortex, synaptogenesis occurs entirely postnatally, allowing study of the mechanisms of synapse formation in vivo. Here we monitored the clustering of GABAA receptors and the scaffolding protein gephyrin at GABAergic postsynaptic sites during rat cerebellar development. We found that GABAA receptors and gephyrin co‐aggregate at nascent synapses in the molecular and Purkinje cell layers with a similar time course. With few exceptions, gephyrin and GABAA receptor subunits clustered selectively in front of presynaptic boutons expressing the vesicular inhibitory amino acid transporter VIAAT and no ectopic localization of these molecules was observed. Surprisingly, gephyrin clusters outlining the cell body of Purkinje cells were transient, and disappeared rapidly at the end of the second postnatal week. The loss of gephyrin from perisomatic synapses was coincident with a significant reduction in the size of GABAA receptor clusters. Furthermore, these changes were accompanied by a developmental decrease in the size of synaptic appositions, as documented by electron microscopy. These findings suggest that gephyrin takes part in the initial assembly of postsynaptic specializations and reveal an unsuspected heterogeneity in the molecular organization of the postsynaptic apparatus at somatic and dendritic synapses of mature Purkinje cells. J. Comp. Neurol. 508:579–591, 2008.

Chronic Administration of the N-Methyl-D-Aspartate Receptor Antagonist Ketamine Improves Rett Syndrome Phenotype.

BACKGROUND Rett syndrome (RTT) is a neurological disorder caused by mutation of the X-linked MECP2 gene, which results in the progressive disruption of excitatory and inhibitory neuronal circuits. To date, there is no effective treatment available for the disorder. Studies conducted in RTT patients and murine models have shown altered expression of N-methyl-D-aspartate receptors (NMDARs). Genetic deletion of the NMDAR subunit, GluN2A, in mice lacking Mecp2 is sufficient to prevent RTT phenotypes, including regression of vision. METHODS We performed a systematic, randomized preclinical trial of chronic administration of low-dose (8 mg/kg, intraperitoneal) ketamine, an NMDAR antagonist, starting either early in development or at the onset of RTT phenotype in Mecp2-null mice. RESULTS Daily exposure to ketamine ameliorated RTT symptoms and extended the life span of treated Mecp2-null mice without adverse side effects. Furthermore, significant improvement was observed in cortical processing and connectivity, which were fully restored to a wild-type level, particularly when treatment was started at the onset of regression. CONCLUSIONS Our findings provide strong evidence that targeting NMDA receptors can be a safe and effective treatment for RTT.

Cell-Specific Regulation of N-Methyl-D-Aspartate Receptor Maturation by Mecp2 in Cortical Circuits.

BACKGROUND Early postnatal experience shapes N-methyl-D-aspartate receptor (NMDAR) subunit composition and kinetics at excitatory synapses onto pyramidal cells; however, little is known about NMDAR maturation onto inhibitory interneurons. METHODS We combined whole-cell patch clamp recordings (n = 440) of NMDAR-mediated currents from layer-4-to-layer-2/3 synapses onto pyramidal and green fluorescent protein labeled parvalbumin-positive (PV) interneurons in visual cortex at three developmental ages (15, 30, and 45 postnatal days) with array tomography three-dimensional reconstructions of NMDAR subunits GluN2A- and GluN2B-positive synapses onto PV cells. RESULTS We show that the trajectory of the NMDAR subunit switch is slower in PV interneurons than in excitatory pyramidal cells in visual cortex. Notably, this differential time course is reversed in the absence of methyl-CpG-binding protein, MECP2, the molecular basis for cognitive decline in Rett syndrome and some cases of autism. Additional genetic reduction of GluN2A subunits, which prevents regression of vision in Mecp2-knockout mice, specifically rescues the accelerated NMDAR maturation in PV cells. CONCLUSIONS We demonstrate 1) the time course of NMDAR maturation is cell-type specific, and 2) a new cell-type specific role for Mecp2 in the development of NMDAR subunit composition. Reducing GluN2A expression in Mecp2-knockout mice, which prevents the decline in visual cortical function, also prevents the premature NMDAR maturation in PV cells. Thus, circuit-based therapies targeting NMDAR subunit composition on PV cells may provide novel treatments for Rett syndrome.

Selective localization of collybistin at a subset of inhibitory synapses in brain circuits

Collybistin is a brain‐specific guanine nucleotide exchange factor (GEF) that is crucial for the postsynaptic accumulation of gephyrin and γ‐aminobutyric acid A receptors (GABAARs) at a specific subset of inhibitory synapses. Our understanding of the in vivo function of collybistin has been hampered by lack of information about the synaptic localization of this protein in brain circuits. Here we describe the subcellular localization of endogenous collybistin by using antibodies raised against distinct molecular domains that should recognize the majority of endogenous collybistin isoforms. We show that collybistin co‐clusters with gephyrin and GABAARs in synaptic puncta and is recruited to postsynaptic specializations early during synapse development. Notably, collybistin is present in only a subset of gephyrin‐positive synapses, with variable co‐localization values in different brain regions. Moreover, collybistin co‐localizes with GABAARs containing the α1, α2, or α3 subunits, arguing against a selective association with specific GABAAR subtypes. Surprisingly, we found that collybistin is expressed only transiently in Purkinje cells, suggesting that in these cerebellar neurons collybistin plays a selective role during the initial assembly of postsynaptic specializations. These data reveal a remarkable heterogeneity in the organization of GABAergic synapses and provide an anatomical basis for interpreting the variable effects caused by disruption of the collybistin gene in human X‐linked intellectual disability and mouse knockout models. J. Comp. Neurol., 2012.